IKZF1 alterations in acute lymphoblastic leukemia: The good, the bad and the ugly

Blood Reviews xxx (xxxx) xxxx

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Blood Reviews journal homepage: www.elsevier.com/locate/blre

Review

IKZF1 alterations in acute lymphoblastic leukemia: The good, the bad and the ugly Stephanie Vairy, Thai Hoa Tran

⁎

Division of Pediatric Hematology-Oncology, Charles-Bruneau Cancer Center, CHU Sainte-Justine, Montréal, Québec, Canada

ARTICLE INFO

ABSTRACT

Keywords: IKZF1 IKAROS Alteration Leukemia Prognosis ALL

Advances in genomics have deepened our understanding of the biology of acute lymphoblastic leukemia (ALL), defined novel molecular leukemia subtypes, discovered new prognostic biomarkers and paved the way to emerging molecularly targeted therapeutic avenues. Since its discovery, IKZF1 has generated significant interest within the leukemia scientific community. IKZF1 plays a critical role in lymphoid development and its alterations cooperate to mediate leukemogenesis. IKZF1 alterations are present in approximately 15% of childhood ALL, rise in prevalence among adults with ALL and become highly enriched within kinase-driven ALL. A cumulating body of literature has highlighted the adverse prognostic impact of IKZF1 alterations in both Philadelphia chromosome (Ph)-negative and Ph-driven ALL. IKZF1 alterations thus emerge as an important prognostic biomarker in ALL. This article aims to provide a state-of-the-art review focusing on the prognostic clinical relevance of IKZF1 alterations in ALL, as well as current and future therapeutic strategies targeting IKZF1-altered ALL.

1. Introduction Acute lymphoblastic leukemia (ALL) is an aggressive hematologic malignancy with an estimated incidence of 0.3% among all new cancer cases in the United States [1]. It represents approximately 20% of all pediatric cancers, being the most prevalent malignancy among patients under the age of 15 [2]. Outcome of pediatric ALL has drastically improved over the past five decades, but 15–20% of children continue to relapse despite modern intensive chemotherapy regimens [2,3]. Results in adults have not kept pace with those of pediatric ALL, with recent 5year overall survival (OS) reaching about 60% by integration of pediatric-inspired chemotherapy protocols for young adults [4,5]. Moreover, this survival rapidly decreases with increasing age [6]. With the advent of next-generation sequencing technologies, the genomic landscape of childhood and adult ALL has been thoroughly investigated and better understood; ALL is thus governed by recurrent chromosomal alterations encompassing gains, losses and rearrangements that confer distinct prognosis. Among the B-lineage ALL (B-ALL)’s “bad players” that have emerged over the years, hypodiploid, intrachromosomal amplification of chromosome 21 (iAMP21), KMT2A-rearranged (KMT2A-r), TCF3-HLF, Philadelphia chromosome-positive (Ph+), and Philadelphia chromosome-like (Ph-like) ALL remain among the ALL subtypes with the worst prognosis [7,8]. Most international protocols

now include them in their risk stratification and incorporate molecular targeted therapy in the relevant subtypes such as Ph + or Ph-like ALL [5]. However, another biomarker has been identified in the 1990s and has consistently demonstrated its prognostic relevance in ALL: IKZF1 alterations. Very few clinical trials have integrated the IKZF1 gene status in their risk stratification algorithm despite increasingly convincing data that have been published on the poor outcomes associated with IKZF1-altered ALL. This article aims to comprehensively review the IKZF1 gene and its function, the spectrum of IKZF1 alterations along with the emphasis on its prognostic impact in pediatric and adult ALL, as well as potential IKZF1-directed therapeutic opportunities. 2. Gene description and function IKZF1 encodes the transcription factor IKAROS, which is a member of the family of zinc finger DNA-binding proteins required for lymphoid lineage ontogeny and homeostasis. The rest of the family comprises IKZF2 (HELIOS), IKZF3 (AIOLOS), IKZF4 (EOS), and IKZF5 (PEGASUS) [9–11]. The IKZF1 gene is mapped on the short arm of chromosome 7 at 7p12.2 [12,13]. It is composed of 8 exons, coding for 519 amino acids. Exons 4 to 6 encode the four N-terminal DNA-binding zinc fingers, while exon 8 harbors the two C-terminal domain zinc fingers necessary for homo- or heterodimerization [14,15]. At least 16 different isoforms

⁎ Corresponding author at: Division of Pediatric Hematology-Oncology, CHU Sainte-Justine, 3175 Côte Sainte-Catherine, Local A-435, Montréal, QC H3T 1C5, Canada. E-mail address: [email protected] (T.H. Tran).

https://doi.org/10.1016/j.blre.2020.100677

0268-960X/ © 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: Stephanie Vairy and Thai Hoa Tran, Blood Reviews, https://doi.org/10.1016/j.blre.2020.100677

Blood Reviews xxx (xxxx) xxxx

S. Vairy and T.H. Tran

can be created by alternative splicing or intragenic deletion; each isoform exerts its respective function during specific stages of lymphocyte development [16–18]. Those isoforms vary in length, particularly in the number of N-terminal zinc fingers, thus influencing their DNA-binding capacity and functional properties [18,19]. Importantly, at least 3 Nterminal zinc fingers are required for DNA-binding [20]. Only a few isoforms contain this requisite for DNA-binding: IK1 to IK3; they are localized in the nucleus [17]. IK4 contains only 2 N-terminal zinc finger motifs. IK5 has only one N-terminal zinc finger, and IK6 is depleted of the 4 N-terminal zinc fingers [20]. Non-DNA-binding isoforms exert a dominant-negative (DN) effect and are mislocalized in the cytoplasm instead of the nucleus [17]. In 1991, Lo and colleagues discovered the protein LYF1 that interacted strongly with TdT, but also at smaller extent with B and T cell promoter sites such as μB (part of μ IgH enhancer), λ5 (CD179b), VPREB (CD179a) and LCK. Moreover, it was expressed in most B and T cell lines at different stages of differentiation. They hypothesized that LYF1 was a regulator of lymphocyte-specific transcription [21]. Later, Georgopoulos et al. described the 1kzf1 gene and its expression during fetal hematopoiesis as well as prior to lymphoid progenitors' development in the mouse [16,22]. Here, Ikzf1 seems to be also expressed throughout the T cell maturation steps, at the later stage of lymphoid development [16,23]. Subsequently, LYF1 was recognized as one of the Ikzf1 isoforms. Ikzf1 and its long isoforms recruit and bind to key genes involving in lymphoid transcription through chromatin-remodelling complexes and epigenetic modification [24,25]. Moreover, IKAROS seems to repress transcription of genes promoting PI3K/AKT signaling pathways and stimulate PI3K inhibition, particularly PIK3CD [26]. Furthermore, by binding to TdT, IKAROS competes with the ELF-1 transcription factor for thymocyte differentiation [27]. It also activates kinase-signaling cascades to support B-cell proliferation and differentiation [28,29]. On the other side, it has repression mechanisms using both histone deacetylase complexes (HDAC) and HDAC-independent system [30,31]. Ubiquitination and sumoylation are other transcription repressor processes regulating IKAROS function [32,33]. Therefore, IKAROS plays a pivotal role in almost every steps of normal lymphoid differentiation [18]. The effect of dominant-negative Ikzf1 mutation on DNA binding mediated by the deletion of exons 3 and 4 in Ikzf1 knock-out mice clearly demonstrated early and isolated arrest in lymphoid development [22]. Moreover, when those mice harboured a germline Ikzf1 heterozygous mutation, they developed a T-cell lymphoproliferative disease, in a timing corresponding to the loss of the remaining Ikzf1 wild-type allele. As mentioned above, this dominant-negative effect appeared to be mediated by elevated levels of mutant non-binding DNA isoforms having less than 3 N-terminal sequences [23]. They affected transcriptional activity by sequestering wild-type isoforms in heterodimers causing altered DNA-binding properties, becoming repressor proteins [19,34]. Mutations in the C-terminal zinc fingers could also be harmful to the formation of homo- and heterodimers of Ikzf1 isoforms, which ultimately led to profound effect on transcription [19,34]. Among all the isoforms, the DN IK6 isoform, which lacked the 4 DNAbinding N-terminal zinc fingers while retaining C-terminal dimerization domains as the result of the deletion of exons 4–7 definitely had the strongest association with oncogenicity [35]. Unlike the other isoforms, IK6 was rarely found in normal hematopoietic cells other than in the lymphoid lineage [36,37]. In BCR-ABL1 plus hypomorphic Ikzf1 transgenic mice, B cell leukemia development was strikingly more accelerated compared to the control groups and to BCR-ABL1/Ikzf1 wildtype [16]. Similarly, in BCR-ABL1 and IK6/Arf −/− transduced mice, disease lineage is shifted to lymphoid leukemia. Based on these observations, Ikzf1 acted as a tumor suppressor and alterations leading to Ikzf1 loss of function represented cooperating genetic events in driving leukemia development [38,39]. Furthermore, the IK6 isoform was also associated with therapy resistance in Ph+ALL [40]. Poorest dasatinib response was observed in mice inoculated with IK6 expression and Arf

loss, and was not due to impaired ABL inhibition neither to the acquisition of ABL1 kinase domain mutation conferring drug resistance [38,39]. Churchman and colleagues described that Ikzf1 alterations conferred stem cell-like properties, induced cell adhesion, led to cellular mislocalization and importantly decreased responsiveness to TKI in their BCR-ABL1 mouse models. 3. Spectrum of IKZF1 alterations in ALL 3.1. Germline mutations In a recent article published by Churchman et al., 28 germline variants of IKZF1 were identified in cases of familial and sporadic childhood B-ALL. Among 4963 children with B-ALL, germline IKZF1 alterations were identified in 45 patients (0,9%), among whom 5 were not singleton. This may suggest the incomplete penetrance of those variants. The described mutations were mostly missense mutations, with 2 nonsense and one frameshift mutations. They were distributed all across the gene, even outside of known DNA-binding or dimerization domains, in contrast to the previously reported somatic IKZF1 variants in B-ALL or germline IKZF1 variants in combined variable immunodeficiency disease, in which IKZF1 mutations arise predominantly within the N-terminal DNA-binding domains. Moreover, they demonstrated the in vitro and in vivo effects of germline IKZF1 alterations on protein mislocalization, cell adhesion and treatment response. A case in this series presented with variable lymphopenia and low-normal IgG levels without overt immunodeficiency [41]. Immunodeficiency such as dys- and hypogammaglobulinemia with auto-immunity has been described with IKZF1 germline mutations, and in patients with hypogammaglobulinemia developing ALL [42,43]. Germline IKZF1 mutations reported in this context are depicted in Fig. 1B-C. 3.2. Somatic mutations The most common type of IKZF1 alteration is focal deletion, resulting in loss-of-function in the majority of cases (Fig. 1B) [44]. In a large Ph+ ALL cohort described by Mullighan et al., the deletions were predominantly mono-allelic, and confined to exons 4–7 (Δ4–7), giving rise to the DN IK6 isoform. Moreover, larger deletions can occur, leading to the formation of IK9 isoform with deletion of exons 2–6 and IK10 isoform with deletion of exons 1–6 [35]. In adult Ph + ALL studies, 37%–64% had IKZF1 deletion of exons 4–7 and 18–20% had deletion of exons 2–7. Of note, 10%–12% had monosomy 7, creating IKAROS haploinsufficiency [44,45]. Other deletions were found but at lower frequencies such as double heterozygous, deletion of the entire gene and GRB1, deletion from promoter to exon 7, and deletion from promoter to exon 1 [44]. In pediatric B-ALL, whole gene and exons 4–7 deletions predominate and altogether account for 62% of the IKZF1mutated cohort. Other deletions were reported, such as deletions involving exons 2–3, exons 2–7, and exons 4–8. Once again, biallelic deletions were only observed in a small subset (2.6%) [46–48]. Previous work published by Mullighan et al. described missense, frameshift and nonsense mutations in their pediatric high-risk (HR) BALL patients (Fig. 1A) [46]. Sequence mutations in the N-terminal zinc fingers at or near residues are expected to have similar effect than that of focal deletions on IKAROS function and have been extensively reviewed elsewhere [13,39,46,49]. Rarely, gene fusions implicating IKZF1 have also been described in pediatric B-ALL, but their functional effects remain largely speculative. Among associated fusion partners, NUTM1, STED5, CDK2, and TRPV2 have been reported [50]. Of note, the type of IKZF1 alterations does not differ between Ph + and Ph-negative ALL patients [51]. Interestingly, the international multicenter study published by Boer and colleagues suggested that the type of IKZF1 deletions might confer differential outcomes. They demonstrated through case-control method that deletion of exons 2–7 and exons 2–8 confer worse prognosis than the more common 2

Blood Reviews xxx (xxxx) xxxx

S. Vairy and T.H. Tran

Fig. 1. Spectrum of germline and somatic IKZF1 alterations in acute lymphoblastic leukemia. A. Summary of IKZF1 mutations reported in the literature on a schematic genomic representation of the IKZF1 gene. N-terminal and C-terminal zinc fingers are represented in green. Each mutation is represented by a line at its specific location, and each type of mutation has a distinct colour. B. Summary of IKZF1 deletions. Each line represents the length of deletion. Arrows are used for deletion extending beyond the limits of the gene. Dashed lines are for homozygous deletions while solid lines are for hemizygous deletions. The red arrow represents IK6 isoform with its loss of exon 4–7, including the 4 N-terminal DNA-binding zinc-fingers and associated with poor prognosis. The orange line is a germline heterozygous deletion in exon 4 and 5. Frequencies (%) of the most common IKZF1 deletions (DEL) in pediatric IKZF1-altered ALL are in black. C. Germline mutations reported in IKZF1. Each mutation is represented by a line at its specific location, and each type of mutation has a distinct colour.

deletions (whole gene deletion and Δ4–7). The only equivocal hazard ratio when compared to wild-type IKZF1 was deletion of exons 4–8, illustrating the variable prognosis associated with different types of IKZF1 deletions [52].

classically associated with a poor prognosis, but its outcome has drastically improved with the advent of tyrosine kinase inhibitor (TKI) in combination with cytotoxic chemotherapy backbone [57,58]. Among Ph + ALL, 70–80% of patients harbor IKZF1 alterations [35,44,59]. This number tends to be higher in adult Ph + ALL. Indeed, reported IKZF1 alterations in pediatric Ph + ALL studies range from 64 to 76,2% [35,48], while they increase to 84% among adult Ph + ALL [60]. Another ALL subtype in which IKZF1 alterations predominate is Phlike ALL. The latter accounts for over 25% of adults with B-ALL [61] and 15% of childhood B-ALL [8,62]. As extensively reviewed elsewhere, Ph-like ALL patients exhibit adverse clinical features, have poor outcomes despite modern chemotherapy regimens, and share a similar gene-expression profile to that of Ph + ALL but lack the canonical BCRABL1 oncoprotein; thus known as Ph-like ALL. Alternatively, the molecular hallmark of Ph-like ALL is characterized by a diverse spectrum of kinase-activating alterations, which can be further divided into the following subgroups: 1) JAK-STAT pathway mutations (CRLF2 rearrangements +/− JAK mutations, JAK2 fusions, EPOR fusions, sequence mutations of SH2B3 and IL7R); 2) ABL-class fusions (ABL1,

4. Prevalence of IKZF1 alterations in ALL In order to better characterize IKZF1 alterations, multiple groups have studied its prevalence and prognostic impact among different ALL subtypes. Overall, IKZF1 alterations average 15% in pediatric Ph-negative B-ALL [35,53]. The prevalence of IKZF1 alterations rises to 30% among pediatric NCI HR B-ALL populations, excluding infant, hypodiploid and Ph + ALL [46,54]. Among adults with ALL, its prevalence increases to 30–50% [53,55,56]. The prevalence of IKZF1 alterations among pediatric and adult Ph-negative and Ph + ALL is summarized in Tables 1 and 2 respectively. IKZF1 alterations are enriched in ALL subtypes associated with poor outcomes, typically in kinase-activating leukemias. Ph + ALL comprises 3–5% of pediatric ALL and over 25% of adult ALL. This subtype is 3

4

AIEOP-BFM 2000 cohort

Indian cohort

ANZHCHOG ALL8 cohort

Stanulla et al., J Clin Oncol [95]

Singh M et al., 2018 Leuk Res [113]

Sutton R et al., 2018 Br J Haematol [114]

COG AALL0622 cohort

Slayton et al., J Clin Oncol [94]

DFCI ALL 05–001 cohort

DFCI ALL 05–001 cohort

Vrooman et al., Blood Adv [83]

Tran et al., Blood Adv [54]

Malaysian-Singapore MS ALL 2003 and MS 2010 cohorts

Non-high-risk Ph-negative BCP-ALL < 18 years old

BCP-ALL including Ph + ALL < 18 years old

Ph-negative BCP-ALL < 18 years old

NCI HR Ph-negative BCPALL < 18 years old

Ph + ALL < 18 years old

BCP-ALL < 18 years old

BCP-ALL including Ph+/Ph-like ALL < 18 years old

BCP-ALL including Ph+/Ph-like ALL < 18 years old

DCOG/ COALL cohorts

Yeoh et al., J Clin Oncol [98]

BCP-ALL < 18 years old

2 Mexican institutions

Ayon-Perez et al., 2019 Cytogenet Genome Res [111] Steeghs EMP et al., 2019 Sci Rep [112]

Patient population

Cooperative groups/trials

Clinical studies from 2009 to 2019

50/475 (11)

28/94 (30) of NCI HR Phnegative BCP-ALL, and 11/16 (69) of patients with kinase-activating fusion. 146/991 (15) among Phnegative BCP-ALL, and 63/991 (6) harbors the IKZF1plus profile. 11/67 (16)

25/44 (56.8)

62/385 (16)

103/515 (20) for the overall BCP-ALL cohort, 13/20 (65) among Ph + ALL, 39/89 (44) among Ph-like ALL, and 4/148 (3) among ETV6RUNX1 B-ALL. 109/685 (15.9) of all BCP-ALL, 24/34 (70.6) among Ph + ALL, and 15/25 (60) among Ph-like ALL.

7/34 (20.6)

Prevalence of IKZF1 alterations (%)

Deletions/ MLPA

Deletions/ MLPA

Deletions/ MLPA

Deletions/ MLPA

Deletions/ Exome sequencing

Deletions (DN: 39%; whole gene deletion: 52%; others: 10%)/ MLPA

Deletions (DN isoforms: 35.8%; whole complete deletion: 27.5%)/ MLPA

Deletions/MLPA

Deletions (but no DN isoforms)/MLPA

Type and/or frequency of IKZF1 alterations/methods of detection

Table 1 Clinical studies assessing the prevalence and prognostic impact of IKZF1 alterations in pediatric B-lineage acute lymphoblastic leukemia.

5-year EFS and OS of IKZF1deleted patients were 45% and 66% compared to 81% and 90% for those with IKZF1 WT, p < .001 and p = .004 5-year EFS was 53% for IKZF1plus patients, 79% for IKZF1-deleted only patients and 87% for IKZF1 WT patients, p < .001 2-year EFS was 56% for IKZF1 and/or CDKN2A deletions compared to 80% for those with neither, p = .05 7-year CIR and EFS for IKZF1deleted patients were 41% and 53% compared to 15% (p < .0001) and 83% (p < .0001) for IKZF1 WT patients respectively.

5-year CIR for IKZF1-deleted on MS 2003 decreased from 30.4% to 13.5% on MS 2010 (p = .05); and the 5-year OS improved from 69.6% to 91.6% (p = .007). 5-year EFS for IKZF1-deleted was 63% vs 88% for IKZF1 WT patients, p < .001 5-year EFS and OS were 52% and 80% for IKZF1-deleted vs 82% vs 100% for IKZF1 WT patients, p = .04 and p = .04 respectively.

5-year CIR for IKZF1-deleted group 30.4% vs 9.0% for IKZF1 WT group (p < .001) 5-year CIR for IKZF1-deleted patients 13.5% vs 4.8% in IKZF1 WT patients; p = .03)

5-year EFS for IKZF1-deleted group 66.3% vs 88.2% for IKZF1 WT group (p = .001)

All patients in complete remission and no relapse.

Prognostic impact of IZKF1 alterations

(continued on next page)

IKZF1 deletion status represents an independent factor for worse RFS. The combination of IKZF1 deletion and P2RY8-CRLF2 in addition to NCI risk group and MRD at Day 33 > 5 × 10−5 predicts a group of patients with the worse outcome.

IKZF1plus prognostic effect varies according to MRD response, refining risk stratification among those with intermediate or high-risk by MRD. Small patient numbers and short followup.

IKZF1 deletion status was identified in multivariable analyses as an adverse prognostic factor for DFS and EFS. IKZF1 deletions still confer inferior outcomes among Ph + ALL patients who achieved early MRD negativity (5year EFS was 50% for IKZF1-deleted vs 83% for non-IKZF1 deleted, p = .04) IKZF1 deletion represents an independent adverse factor of poor outcomes, irrespective of the presence of kinase-activating fusion.

MS 2010 was the first prospective clinical trial incorporating IKZF1 status in risk stratification. Treatment intensification for those with IKZF1 deletion in MS 2010 study protocol has significantly improved their outcomes compared to those treated on the predecessor study MS 2003.

IKZF1 deletion was predictive of unfavorable outcome on DCOG-ALL10, independently of early treatment response by MRD.

Small sample size and no long-term data available.

Comments

S. Vairy and T.H. Tran

Blood Reviews xxx (xxxx) xxxx

BFM 95 cohort

International consortium including DCOG, BFM, COALL, CCLG, ANZCHOG, AIEOP, PPLLSG, CPH and BPHOP cohorts Mexican cohort

EORTC-CLG 58951 cohort

NOPHO cohort

Ponte di Legno and EsPhALL cohorts

NOPHO ALL 1992, 2000 or 2008 cohorts

Japanese cohort

Japanese JACLS ALL02 cohort

Ponte di Legno and EsPhALL cohorts

BFM ALL 2000 cohort

Hinze L et al., 2017 Leukemia [115]

Boer et al., Leukemia [52]

Clappier E et al., 2015 Leukemia [117]

Olsson et al., Br J Haematol [76]

Lana T et al., Leukemia, 2015

Genes Chromosomes Cancer [49,76]

Hanada I et al., 2014 Genes Chromosomes Cancer [118]

Yano M et al., 2014 Genes Chromosomes Cancer [119]

van der Veer et al., Blood [59]

Zaliova M et al., 2014 Leukemia [120]

Reyes Leon A et al.,2015 PLoS One [116]

Cooperative groups/trials

Clinical studies from 2009 to 2019

Table 1 (continued)

5 ERG-deleted BCP-ALL < 18 years old

Ph + ALL

Ph-negative BCP-ALL < 15 years old

DS-ALL < 20 years old

BCP-ALL < 18 years old

Ph + ALL < 18 years old

Ph-negative BCP- ALL < 18 years old

Ph-negative BCP-ALL < 18 years old

BCP-ALL < 18 years old

BCP-ALL between 1 and 19 years old

Ph-negative BCP-ALL < 18 years old

Patient population

26/60 (43)

126/191 (66)

25/167 (15)

8/140 (5.7) of BCP-ALL had IKZF1 sequence mutations; 27/140 (19.3) had either IKZF1 mutations and/or deletions 8/32 (25)

196/295 (66) with IKZF1 deletions; 12/295 (4) with IKZF1 mutations

50/334 (15)

179/1223 (14.6)

88/134 (66) IKZF1deleted cases had rare IKZF1 deletions comprising at least exon 2. 23/99 (23.2)

80/655 (12.2)

Prevalence of IKZF1 alterations (%)

Deletions (haploinsufficient: 36.5%; DN: 52.4%; miscellaneous: 11.1%)/ MLPA Deletions/ MLPA

Deletions/ MLPA

Deletions/ MLPA

Deletions and mutations/ SNP and Targeted deep sequencing

Deletions and mutations/ MLPA and NGS

Deletions (focal deletions: 58%; nonfocal deletions: 42%)/ SNP and MLPA

Deletions (whole-gene deletions: 37%; DN: 35%; rare intrageneic deletions: 28%)/ Multiplex PCR and MLPA

IK6 and IK8 isoforms/ RT-PCR

Rare IKZF1 variants (rare IKZF1 deletions, exluding whole gene and exons 4–7 deletions)/ MLPA

Deletions/ PCR

Type and/or frequency of IKZF1 alterations/methods of detection

4-year DFS of IKZF1-deleted patients was 30.0% vs 57.5% for IKZF1 WT (p = .013) 5-year EFS of ERG-deleted patients was higher in IKZF1deleted patients, 85% vs 64% for IKZF1 WT (p = .074)

5-year OS for IKZF1-deleted patients was 41.7% vs 83.8% for IKZF1 WT (p = .041). EFS was not significantly affected. 5-year OS for IKZF1-deleted patients was 41.7% vs 83.8%; p = .041)

4-year DFS of IKZF1-altered (deletion + mutation) patients was 28.1% vs 64.3% for IKZF1 WT (p = .0036) 10-year EFS for IKZF1-altered patients was 49% vs 72% for IKZF1 WT (p = .008)

10-year EFS for IKZF1-deleted patients was 60% vs 83% for IKZF1 WT (p < .001)

Expression of IK6 and/or IK8 isoforms was not independently associated with relapse or death. The 8-year EFS, OS and CIR for IKZF1-deleted patients was 67.7%, 86.7% and 25.7% vs 86.5% (p < .001), 92.4% (p = .035) and 10.8% (p < .001) for IKZF1 WT

All variants of rare IKZF1 deletions are associated with unfavorable prognosis in pediatric B-ALL.

5-year EFS of IKZF1-deleted patients was 0.66 vs 0.82 for IKZF1 WT patients (p = .001)

Prognostic impact of IZKF1 alterations

(continued on next page)

IKZF1 deletion but not CRLF2 overexpression was an independent prognostic factor for OS in multivariate analysis. IKZF1 deletion was an independent unfavorable prognostic factor for goodrisk Ph + ALL patients only. ERG deletion attenuated the negative impact of IKZF1 deletions in pediatric ALL.

In contrast to IKZF1 deletion, IKZF1 mutation does not seem to be a significant predictor of relapse, unless associated with concomitant IKZF1 deletion.

Among IKZF1-deleted patients randomized for vincristine-steroid pulses during maintenance, those receiving pulses had a significantly higher 8-year DFS (93.3% vs 42.1%; p < .001). IKZF1 deletion was an independent prognostic factor for risk of relapse. Cooccurrence of IKZF1 deletions and P2RY8-CRLF2 increased the risk of relapse (75% vs 30% for cases with IKZF1-deleted alone; p = .045).

Addition of vincristine-dexamethasone pulses during maintenance therapy in patients with IKZF1 deletion did not improve outcome. The outcome of the rare IKZF1 variants was equal or worse compared to that of the more common IKZF1 del 4–7 and del 1–8.

Comments

S. Vairy and T.H. Tran

Blood Reviews xxx (xxxx) xxxx

6

JCCLSG ALL04 cohort

AEIOP-BFM 2000 cohort

NOPHO cohorts

Yamashita et al., Pediatr Blood Cancer [78]

Palmi C et al.,2013 Haematologica [121]

Ofverholm I et al., 2013 Leukemia [122]

BCP-ALL < 18 years old

Ph-negative BCP-ALL < 18 years old

BCP-ALL < 18 years old

BCP-ALL < 18 years old

ALL-REZ-BFM-2002 cohort

DCOG and UK ALL cohorts

COG P9905 and P9906 cohorts

Taiwanese TPOG-ALL-93, 97 and 2002 cohorts

DCOG ALL9 cohort

Krentz S et al., 2013 Leukemia [124]

Buitenkamp et al., Leukemia [64]

Chen et al., Blood [81]

Yang YL et al., 2011 Cancer Sci [125]

Waanders et al., Leukemia [79]

BCP-ALL < 18 years old

BCP-ALL

Ph-negative BCP-ALL

DS-ALL < 18 years old

Relapsed BCP-ALL < 18 years old

BCP-ALL < 18 years old

NOPHO ALL 1992, 2000 and 2008 cohorts

Olsson et al., Leukemia [74]

NCI HR BCP-ALL < 18 years old

ALL-BFM-2000 cohort

Japanese Chilhood Leukemia Study ALL02

Asai et al., Cancer Med [80]

Ph-negative BCP-ALL < 18 years old

Ph-negative BCP-ALL < 18 years old

DCOG/ COALL cohorts

van der Veer et al., Blood [77]

ERG-deleted BCP-ALL < 18 years old

Patient population

IC-BFM-2002 and ALLBFM-2000 cohorts

EORTC-CLG 58951 cohort

Clappier et al., Leukemia [67]

Volejnikova J et al., 2013 Pediatr Blood Cancer [123] Dorge et al., Haematologica [47]

Cooperative groups/trials

Clinical studies from 2009 to 2019

Table 1 (continued)

20/131 (15.3)

26/242 (10.7)

157/866 (18.1) had IKZF1 deletions; 9/774 (1.2) had IKZF1 mutation

12/119 (29)

68/204 (33.3)

84/694 (12)

14/206 (7)

19/116 (16)

54/410 (13.2)

22/177 (12)

30/184 (16)

19/202 (9.4)

136/857 (16)

12/29 (41.4)

Prevalence of IKZF1 alterations (%)

Deletions and mutations/ Multiplex PCR and Capillary electrophoresis Deletions and mutations/ MLPA and Sanger sequencing

Deletions and mutations/Oligonucleotide array and RT-PCR

Deletions/ CGH and MLPA

Deletions (whole-gene deletion: 35%; DN: 42%; deletion exons 2–7: 7%)/ MLPA Deletions/ MLPA

Deletions/ MLPA

Deletions (whole-gene deletion: 7%; DN: 17%; others: 30%)/ MLPA Deletions (DN: 26%)/ MLPA

Deletions/ MLPA

Deletions/ SNP

Deletions (whole-gene deletions: 40.4%; DN: 26.5%; deletions exons 2–7: 9.6%; deletions exons 4–8:7.3%; deletions exons 2–3: 8.1%; deletions exons 2–8: 4.4%; other deletions: 3.7%/ MLPA Deletions/ MLPA

Deletions/ MLPA

Type and/or frequency of IKZF1 alterations/methods of detection

5-year EFS of IKZF1-deleted patients was 15.4% vs 77.9% for IKZF1 WT (p < .0001) 9-year RFS for IKZF1-altered patients among MRD-M group was 27% vs 96% for IKZF1 WT (p < .001)

5-year EFS for IKZF1-deleted patients was 62.7% vs 88.8% for IKZF1 WT (p = .001) 10-year EFS for IKZF1-deleted patients was 45% vs 80% for IKZF1 WT (p ≤.001) 4-year EFS for IKZF1-deleted patients was 68.2% vs 85.2% for IKZF1 WT (p = .04) 5-year EFS for IKZF1-deleted patients was 70.2% vs 85.2% for IKZF1 WT (p = .007) Both EFS and OS were significantly reduced in IKZF1-deleted patients versus IKZF1 WT (p = .02 and p = .001) 5-year EFS for IKZF1-deleted patients was 41% vs 86% for IKZF1 WT (p < .0001) 5-year EFS for IKZF1-deleted patients was 69% vs 85% for IKZF1 WT (p < .0001) Unfavorable pEFS for IKZF1deleted patients was 30% vs 51% for IKZF1 WT (p = .002) 6-year EFS for IKZF1-deleted patients was 31% vs 75% for IKZF1 WT (p < .0001) RFS was significantly poorer for IKZF1-deleted NCI HR B-ALL, but not NCI SR B-ALL.

8-year EFS of ERG-deleted patients with IKZF1 deletion was 87.5% vs 88.2% for IKZF1 WT (p = .63) 5-year CIR for IKZF1-deleted patients was 34% vs 15% in IKZF1 WT (p ≤.001)

Prognostic impact of IZKF1 alterations

(continued on next page)

IKZF1 deletion was an independent poor prognostic factor in multivariate analysis. IKZF1 deletion was the strongest independent risk factor for poor outcomes in multivariate analysis. IKZF1 deletion was an independent poor prognostic factor in multivariate analysis for the full cohort of BCP-ALL, but did not retain significance among NCI HR B-ALL cases. IKZF1 deletion was an independent poor prognostic factor in multivariate analysis. The integration of IKZF1 deletion status and MRD accurately predicts 79% of relapses in pediatric B-ALL.

IKZF1 deletions and MRD on Day 15 retained independent prognostic significance in multivariate analysis. IKZF1 deletion was an independent prognostic factor.

IKZF1 deletion retained prognostic significance on EFS in multivariate analysis. IKZF1 deletion was an independently risk factor for poor EFS and OS.

IKZF1 deletion was the strongest independent risk factor for poor EFS in multivariate analysis. IKZF1 deletion not statistically significant in multivariate analysis.

IKZF1 deletion was an independent prognostic factor of poor outcomes

The IKZF1 deletion and BCR-ABL-like signature represent independent adverse factors for outcome

Comments

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Deletions (whole-gene deletion: 24%; DN: 30%) and mutations (9%)/ SNP 67/221 (30.3) BCP-ALL COG P9906 and St.Jude ALL cohorts Mullighan et al., N Engl J Med [46]

BCP-ALL: B-cell progenitor acute lymphoblastic leukemia; DN: Dominant-negative; MLPA: Multiplex ligation-dependent probe amplification; DCOG: Dutch Chilhood Oncology Group; COALL: Childhood Oncology Acute Lymphoblastic Leukemia; Ph + ALL: Philadelphia chromosome-positive acute lymphoblastic leukemia; Ph-like ALL: Philadelphia chromosome-like acute lymphoblastic leukemia; EFS: event-free survival; WT: wild-type; CIR: cumulative incidence of relapse; MRD: minimal residual disease; DFCI: Dana-Farber Cancer Institute; DFS: disease-free survival; NCI HR: National Cancer Institute – High Risk; COG: Children's Oncology Group; OS: overall survival; AIEOP-BFM: Associazione Italiana di Ematologia e Oncologia Pediatrica-Berlin-Frankfurt-Muenster; ANZHCOG: Australian &New Zealand Childrens Hematology-Oncology Group; CCLG: Children's Cancer and Leukemia Group; PPLLSG: Polish Pediatric Leukemia/Lymphoma Study Group, CPH: Czech Pediatric Hematology; BPHOP: Brazilian Pediatric Hematology-Oncology Program; EORTC-CLG: European Organisation for Research and Treatment of Cancer-Children's Leukemia Group; NOPHO: Nordic Society of Pediatric Hematology and Oncology; JACLS: Japanese Association of Childhood Leukemia Study; DS-ALL: Down syndrome – Acute Lymphoblastic Leukemia; CGH: comparative genomic hybridization; TPOG: Taiwan Pediatric Oncology Group; RT-PCR: reverse transcription polymerase chain reaction; SNP: single nucleotide polymorphism.

IKZF1 deletion was the strongest independent adverse factor in multivariate analysis. First landmark study demonstrating that IKZF1 deletion was an independent adverse prognostic factor in multivariate analysis. 8-year RFS of IKZF1-deleted patients was 39% vs 89% for IKZF1 WT (p < .001) 5-year EFS of IKZF1-altered patients was 25.9% vs 72.8% for IKZF1 WT (p < .0001) Deletions and mutations/MLPA, Sanger sequencing and SNP Relapsed BCP-ALL < 18 years old DCOG cohorts Kuiper RP et al., 2010 Leukemia [126]

11/24 (45.8)

Prognostic impact of IZKF1 alterations Cooperative groups/trials Clinical studies from 2009 to 2019

Table 1 (continued)

Patient population

Prevalence of IKZF1 alterations (%)

Type and/or frequency of IKZF1 alterations/methods of detection

Comments

S. Vairy and T.H. Tran

ABL2, CSF1R, PDGFRA/B); 3) Ras pathway mutations; and 4) rare kinase fusions (NTRK3, DGKH, PTK2B, LYN) [8]. Roberts and colleagues reported the prevalence of IKZF1 alterations to be 73% among adult Phlike ALL, compared to 27–68% of childhood Ph-like ALL [8,61–63]. IKZF1 alterations were also more common among Ph-like ALL patients harboring kinase fusions compared to those with sequence mutations (78% vs 33%; p < .001) [8]. Down syndrome–associated ALL (DS-ALL) also has a high prevalence of CRLF2 rearrangements (62%) and IKZF1 alterations (35%) [64]. A single institution study reported a high likelihood of DS patients among Ph-like ALL, which could account for the high prevalence of CRLF2 and IKZF1 alterations in DS-ALL [62]. However, it is not known whether all cases of DS-ALL harboring CRLF2 rearrangements constitute Ph-like ALL cases defined by kinase-activated expression signatures. Among favourable ALL subtypes, the prevalence of IKZF1 alterations is lower. It accounts for 3–6% of ETV6-RUNX1 cases [47,65]. Intragenic ERG deletion was initially identified by Mullighan et al. then later reported by other groups to occur in approximately 3–8% of B-ALL [50,53,66,67]. ERG deregulation represents the hallmark of this subtype, through intragenic ERG deletions primarily, but also through expression of aberrant ERG transcripts [68]. Among patients with ERGderegulated ALL subtype, 38–43% of them also harbored concomitant IKZF1 deletions, predominantly the Δ4–7. ERG-deregulated B-ALL is characterized by a unique gene expression profile and is exclusively associated with DUX4 rearrangements as secondary abnormality [50,68,69]. Regarding T-ALL, IKZF1 mutations are less prevalent with rates ranging between 4 and 5% [35,53,70]. Kastner et al. previously reviewed this gene-phenotype association but the prevalence was extremely variable [71]. Regarding early pre-T-cell precursor ALL, one study reported the prevalence rate of 13% [72]. 5. The prognostic impact of IKZF1 alterations in pediatric and adult Ph-negative ALL IKZF1 alterations are enriched in HR ALL subtypes such as Ph + ALL and Ph-like ALL, which are notoriously known to confer poor outcomes. Adverse clinical risk factors such as high white blood cell counts, and older age are often associated with IKZF1-deleted cases [48,73,74]. IKZF1 alterations are frequently associated with elevated end-of-induction minimal residual disease (MRD) levels in pediatric HR B-ALL and consequently, higher cumulative incidence of relapse [46,47]. In several studies, including a meta-analysis of 15 studies [75], IKZF1 deletions have been consistently reported as an independent adverse risk factor, even after adjusting for the above clinical features and leukemia phenotype [45–47,63,74,76–78]. The prognostic significance of IKZF1 alterations in ALL will be discussed below. In spite of the lower prevalence of IKZF1 alterations in Ph-negative ALL as compared to that in Ph + or Ph-like ALL, the negative prognostic factor of IKZF1 alterations in Ph-negative ALL still retained significance. The adverse outcome associated with IKZF1 alterations in Phnegative ALL was first described by Mullighan and colleagues in 2009 in a cohort of HR ALL patients enrolled on Children's Oncology Group (COG) and St. Jude Children's Research Hospital (SJCRH) trials [46]. Both deletion and mutation of IKZF1 were significantly associated with an increased risk of relapse and adverse events, which represented an independent negative predictor of outcomes after adjusting for age, leucocyte count at diagnosis and cytogenetic subtype [46]. This seminal study also highlighted the similarity in the gene expression profile of IKZF1-deleted HR B-ALL to that of Ph + ALL, which led to the discovery of Ph-like ALL [46]. Subsequently, other international consortia replicated similar findings. The Dutch Childhood Oncology Group (DCOG) was the first to demonstrate that the integration of IKZF1 status and MRD response accurately predicted nearly 80% of relapses with a 93% specificity within their DCOG-ALL9 study cohort [79]. IKZF1 alteration status had the most prognostic impact in the MRD-Medium 7

Chinese singlecenter cohort

Chinese singlecenter cohort

Chinese ChiCTRTNRC-09000309 trial

Tang S et al., 2019 Bone Marrow Transplant [127]

Fang et al., Oncol Lett [91]

Li et al., Leuk Lymphoma [93]

8

Adult BCP-ALL

Fang Q et al., 2017 Leuk Lymphoma [130]

Chinese ChiCTRTRC-00000397 cohort

Adult BCP-ALL

77/118 (65.3) among BCPALL; 35/42 (83.3) among Ph + ALL vs 42/76 (55.3) among Ph-negative ALL

28/70 (40) among BCP-ALL and 9/17 (53) among Ph + ALL cases.

128/482 (27)

33/66 (50)

Adult Ph + ALL

Adult Ph-negative BCP-ALL > 16 years old

28/69 (40.6) among BCPALL; 72.4% among Ph + ALL and 17.5% in Phnegative B-ALL.

80/164 (48)

97/116 (84)

Prevalence of IKZF1 alterations (%)

Adult BCP-ALL

Adult BCP-ALL

Adult Ph + ALL

Patient population

Patel S et al., 2017 Leuk Res [129]

GMALL 06/99 and 07/03 cohorts

GIMEMA trials

Fedullo et al., Haematologica [60]

Kobitzsch B et al., 2017 Haematologica [128]

Cooperative groups/Trials

Clinical studies from 2009 to 2019

Deletions/ RT-PCR

Deletions/ SNP arrays

Deletions (12% had loss-offunction deletions, 10% had DN deletions, and 5% had both types)/ PCR

Deletions (DN: 42.5% and IK10 isoforms: 7.5%)/ RT-PCR

Deletions (DN: 42.9%, whole gene deletion: 7.1%)/ MLPA

Deletions (DN: 29.8%; haploinsufficient or whole gene deletion: 57.7%; miscellaneous; 11.3%)/ CytoScan HD Array and MLPA Mutations/ Capillary electrophoresis

Type and/or frequency of IKZF1 alterations/Methods of detection

Worse outcomes were for IKZF1-deleted patients who did not achieve deep MRD response after 3 months. There was a non-significant trend towards inferior OS for patients with any type of IKZF1 deletion (0.46 vs 0.59; p = .06). Patients with loss-of-function deletions had a reduced OS (0.37 vs 0.59; p = .0012), while dominant-negative deletions had no effect on OS (0.54 vs 0.56; p = .95). The CDK2NA/B plus IKZF1 co-deletion was associated in univariate analyses with a significantly worse OS than having only one or neither of these deletions (both vs all others: p = .0077, both vs IKZF1 only: p = .038, both vs CDKN2A only: p = .061 (p = .052 for log-rank test), both vs neither deleted: p = .037). 3-year RFS and OS for patients with IKZF1 deletions were 42.2% and 43.7% vs 63.3% (p = .015) and 63.6% (p = .026) for those without IKZF1 deletions.

2-year RFS for IKZF1-deleted group was 34.3% vs 55.6%; p = .029 IKZF1-deleted patients had a worse DFS (median 4 vs 20 months; p = .007) and OS (median 14 vs 37 months; p = .004).

3-year LFS was significantly higher in IKZF1-mutated patients in HSCT group vs no-HSCT group (54.4% vs 21.7%;p = .016) No statistical significance was seen in outcomes among Ph-/Ph + patients with or without IKZF1 deletion.

3-year DFS for DN IKZF1 isoform 23.3% vs 53.3% for IKZF1 WT; p = .016. 3-year LFS was significantly lower in IKZF1+/Ph + vs IKZF1+/Ph- vs IKZF1-/ Ph- after chemotherapy (19.0% vs 37.9% vs 41.9%; p = .047 and p = .017, respectively)

No significant DFS difference between IKZF1-deleted vs IKZF1 WT groups.

Prognostic impact of IZKF1 alterations

Table 2 Clinical studies assessing the prevalence and prognostic impact of IKZF1 alterations in adult B-lineage acute lymphoblastic leukemia.

(continued on next page)

IKZF1 deletions retained independent prognostic significance for OS and RFS in B-ALL. Concomitant IKZF1 deletion and CRLF2 overexpression confer worse prognosis.

Combining IKZF1 deletion status and early MRD response enable better risk stratification.

Study was limited by small sample size and short follow-up (2-year).

Additional deletions of IKZF1 + CDKN2A ± PAX5 (46.4% of cases) had a worse 3-year DFS and OS compared to IKZF1-deleted only group (24.9% vs 43.3%; p = .026; 40.2% vs 62.6%; p = .02, respectively). IKZF1 mutation confers an independent factor of poor prognosis in adult B-ALL receiving chemotherapy only and HSCT significantly improved LFS and OS of IKZF1-mutated patients.

Comments

S. Vairy and T.H. Tran

Blood Reviews xxx (xxxx) xxxx

CALGB 10001 cohort

Single-center cohort in Northern India

PETHEMA cohorts

Korean cohort

DeBoer R et al., 2016 Leuk Lymphoma [132]

Gupta SK et al., 2016 Leuk Res [133]

Ribera et al., Cancer [89]

Kim M et al., 2015 Bone Marrow Transplant [134] Beldjord K et al., 2014 Blood [135]

9

GIMEMA cohorts

Western Australia

Adult Ph + ALL from 16 to 64 years old Ph-negative BCP-ALL from 15 to 59 years old Adult Ph-negative BCP-ALL Adult Ph + ALL

Adolescent and adult BCP-ALL between 15 and 74 years old

Pediatric and adult BCP-ALL from 1 to 67 years old

Adult Ph + ALL 15–60 years old

Adolescent and adult BCP-ALL > 14 years old

Patient population

52/83 (63)

15/51 (29)

69/216 (31.9)

29/101 (28.7) among BCPALL; 53% in Ph + ALL, 24% in Ph-negative ALL, 47% in adult and 24% in pediatric cases. 49/142 (35) among pre-B ALL; 35/115 (30) among Ph-negative ALL and 14/27 (52) in Ph + ALL. 93/118 (78.8)

22/28 (79)

68/162 (42) among BCPALL; 75% among Ph + ALL vs 25% in Ph-negative ALL

Prevalence of IKZF1 alterations (%)

Deletions (DN: 41%; whole-gene deletion: 22%)/ MLPA and multiplex PCR Deletions/ FISH Deletions (DN:37%; deletion exons 2–7: 20%)/ SNP

Deletions/ MLPA

Deletions/ MLPA

Deletions (haploinsufficiency: 66%; DN:28%; others: 7%)/ MLPA

Deletions/ SNP

Deletions/ RT-PCR and MLPA

Type and/or frequency of IKZF1 alterations/Methods of detection

3-year EFS of IKZF1-deleted patients was 15% vs 41% for IKZF1 WT CIR for IKZF1-deleted patients was 69.1% vs 40.4% for IKZF1 WT (p = .0103)

The 5-year CIR and OS were 70% and 29% for IKZF1-deleted patients vs 47% (p = .019) and 50% (p = .023) for IKZF1 WT IKZF1 deletion was significantly associated with MRD kinetics, but not statistically significant for CIR. 5-year CIR for IKZF1-deleted patients was 53.9% vs 28.6% in IKZF1 WT.

5-year CIR was 55% for IKZF1-deleted patients vs 25% for those without IKZF1 deletions (p = .004). 5-year survival was 48% vs 75% respectively (p = .012) Trend towards inferior DFS for IKZF1deleted patients vs IKZF1 WT (13.2 vs 53.1 months; p = .18) Induction failure rate was higher in IKZF1deleted vs IKZF1 WT (42% vs 9%; p = .002)

Prognostic impact of IZKF1 alterations

IKZF1 deletion was an independent poor prognostic factor for DFS in multivariate analysis.

IKZF1 deletion was not found to be an independent prognostic factor in multivariate analysis.

IKZF1 deletion was an independent prognostic factor for increased CIR in the whole BCP-ALL group, but not in Ph-negative BCP-ALL group.

Combination of the presence of IKZF1 deletion and p210 transcript confers significantly worse DFS in multivariate analysis.

IKZF1 deletion was independently associated with higher risks of relapse, treatment failure and death.

Comments

GIMEMA: Gruppo Italiano Malattie EMatologiche dell'Adulto; Ph + ALL: Philadelphia chromosome-positive acute lymphoblastic leukemia; DN: dominant-negative; HD: high density; MLPA: multiplex ligation-dependent probe amplification; WT: wild-type; DFS: disease-free survival; OS: overall survival; BCP-ALL: B-cell progenitor acute lymphoblastic leukemia; LFS: leukemia-free survival; HSCT: hematopoietic stem cell transplantation; RFS: relapse-free survival; ChiCTR: Chinese Clinical Trial Registry; RT-PCR: reverse transcription – polymerase chain reaction; MRD: minimal residual disease; GMALL: German Multicenter Study Group for Adult ALL; CIR: cumulative incidence of relapse; CALGB: Cancer and Leukemia Group B; SNP: single nucleotide polymorphism; PETHEMA: Programa Español de Tratamientos en Hematología; GRAALL:; FISH: fluorescence in situ hybridization.

O'Reilly J et al., 2013 Pathology [136] Martinelli et al., J Clin Oncol [45]

Chinese singlecenter cohort

Yao QM et al., 2016 BMC Cancer [131]

GRAALL-2003 and 2005 cohorts

Cooperative groups/Trials

Clinical studies from 2009 to 2019

Table 2 (continued)

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group in which almost half of the relapses occurred [79]. In children with Ph-negative ALL treated on the BFM-2000 protocol, the 5-year event-free survival (EFS) was 69% for IKZF1-altered vs 85% for the nonIKZF1-altered group, mainly due to a higher incidence of relapse (21% vs 10%) [47]. Among the large NOPHO ALL 1992 cohort, inferior 10year EFS and OS were observed in patients with IKZF1 alteration in multivariate analysis. Among patients who relapsed, 39% were IKZF1deleted vs 10% with wild-type IKZF1 [74]. Similar poor outcomes in IKZF1-altered patients were observed in the Japanese pediatric Ph-negative B-ALL cohort [80]. IKZF1 deletion remained an adverse prognostic factor even for those who showed a good and early response to prednisolone. In another pediatric Japanese Ph-negative ALL cohort, patients with IKZF1 deletion had significantly lower 4-year EFS compared to those without IKZF1 deletion (68.2% vs 85.2%; p = .04). However, the negative impact of IKZF1 deletion on outcomes was only statistically significant among NCI HR patients (58.3% vs 87%; p = .02) vs NCI SR patients (80% vs 84.4%; p = .75) [78]. These findings from the Japanese cohort mirrored those of a large COG cohort comprising 1061 NCI SR and HR B-ALL patients. Within the entire cohort, NCI risk status, MRD, CRLF2 overexpression and IKZF1 alterations represented the four variables retaining independent prognostic significance in multivariate analysis [81]. However, IKZF1 alterations only conferred poorer relapse-free survival (RFS) in NCI HR but not in SR cases. MRD remained the only independent adverse predictor of RFS in both SR and HR B-ALL patients when stratified by NCI risk group in COG studies [82]. In contrast, IKZF1 deletion was associated with inferior 5-year EFS (63% vs 88%; p = .001) and a higher 5-year cumulative incidence of relapse (CIR) even among patients who achieved MRD negativity (24% vs 8%; p = .001) as reported in the Dana-Farber Cancer Institute (DFCI) ALL Consortium Protocol 05–001. In this study, age ≥ 15 years, WBC ≥ 50 × 109/L, IKZF1 deletion and MRD ≥10−4 were associated with inferior outcome in multivariable analysis [83]. Furthermore, the United Kingdom (UK) ALL consortium developed an integrated genetic classification combining conventional cytogenetic and copy number alterations (CNA) data to reclassify over one thousand B-ALL patients into 2 novel risk groups: Genetic – Good Risk (Group A) and Genetic – Poor Risk (Group B). Group B patients had significantly higher incidence of IKZF1 (~40%), PAX5 (~45%) or CDKN2A/B (~70%) compared to ~80% of group A patients who had none of these deletions. The 5-year EFS and OS of group A patients were significantly better than that of group B patients: 94% vs 79% (p < .001) and 97% vs 83% (p < .001) respectively. Among the 4 variables (genetic risk, MRD, age and white count at diagnosis), only genetic risk and MRD retained statistical significance for outcomes in multivariate analysis. Of note, the clinical features, biologic characteristics and outcomes of group B patients in the UK ALL cohort resemble those of the BCR-ABL1-like patient subsets reported by the Dutch group. The prognostic impact of the UK integrated genetic classifier has been validated in a large international study and remained significant across the 12 participating study groups [84]. In another study comprising 533 pediatric B-ALL from the DCOG, German Cooperative ALL and Australian and New Zealand Children's Hematology and Oncology Group, co-occurrence of IKZF1 and BTG1 deletions was associated with an increased risk of relapse and hence, inferior outcomes compared to sole IKZF1-deleted patients. However, outcomes were not significantly different in cases of BTG1 deletions alone compared to those wild-type BTG1 or IKZF1, suggesting that BTG1 deletions enhanced the adverse impact of IKZF1 deletions in B-ALL. In mice models, BTG1 cooperated with IKZF1 to induce leukemogenesis and Btg1−/−;Ikzf1 −/− mice exhibited enhanced glucocorticoid (GC) resistance [85]. Previous work from the same group had also shown that IKZF1 deletions mediated GC resistance in mice models [86]. In children with DS-ALL and IKZF1 deletions, the 6-year EFS was significantly lower at 45% vs 95% for those without IKZF1 deletion (p = .002) [64]. Among adults with Ph-negative B-ALL, the negative impact of IKZF1 deletion on outcomes has been described in several studies. In a meta-

analysis comprising 8 studies with a total of 1008 adult B-ALL patients, IKZF1 deletion was found to be a negative predictor of OS, EFS, diseasefree survival (DFS), relapse- and progression-free survival. However, the independent adverse prognostic significance of IKZF1 deletion only prevailed among Ph-negative B-ALL patients rather than those with Ph + ALL. The relatively small number of eligible studies, the absence of prospective randomized clinical trials, the inconsistent outcome definitions and the heterogeneous treatment protocols among the eligible studies limit the generalization of this meta-analysis [87]. Other studies involving adult Ph-negative B-ALL did not confirm the independent adverse factor of IKZF1 deletions to predict relapse in Ph-negative BALL [88,89]. In a cohort of 128 adolescents and adults with Ph-negative B-ALL treated on the Spanish Programa Espanol de Tratamientos en Hematologia (PETHEMA) protocols, deletions of IKZF1, PAX5 and CDKN2A/B were identified in 30%, 38% and 44% respectively. Although IKZF1 partial gene deletions were significantly associated with higher cumulative incidence of relapse (CIR) rates, co-deletions of IKZF1 and CDKN2A/B conferred the worse outcome, but only CDKN2A/ B deletions remained independently prognostic when considered with end-of-induction MRD in multivariable analysis [90]. Other adult studies have also demonstrated the additive prognostic impact of the cooccurrence of IKZF1 deletions with other CNAs compared to isolated IKZF1 deletion [90,91]. Finally, it should be emphasized that DUX4 rearrangements and ERG deletion should be ruled out in Ph-negative B-ALL with IKZF1 alterations since they confer favourable outcomes inspite of the presence of IKZF1 alterations. Despite the poor prognostic risk associated with IKZF1 alterations in the majority of ALL subtypes, this negative effect of IKZF1 deletion can be mitigated by the presence of ERG/DUX deregulation. Clappier et al. have showed the favourable outcomes of B-ALL patients with ERG deletion with 8-year EFS and OS of 86.4% and 95.6% respectively, even when associated with frequent IKZF1 deletions [67]. Subsequently, several other groups replicated the very good outcomes of a cohort of B-ALL patients characterized by cryptic DUX4 rearrangements, ERG deletion and IKZF1 deletion [50]. Thus, the effect of IKZF1 is pleotrophic and is positively influenced by the presence of DUX4/ERG deregulation; the latter rather represents the exception to the rule of poor prognosis associated with IKZF1 alterations [68,69,92]. Another example of favourable outcomes associated with IKZF1 alterations can be found within the ETV6-RUNX1-like subtype. The latter comprises 1–3% of childhood B-ALL and is characterized by a gene expression profile similar to that of ETV6-RUNX1 despite the absence of this fusion gene. High frequency of IKZF1 alterations, including IKZF1 fusions, has been described within this subtype. Only 2 patients relapsed among 10 ETV6-RUNX1-like cases, which suggest that the frequent IKZF1 alterations did not confer dismal prognosis within this novel subtype [50]. 6. The prognostic impact of IKZF1 alterations in pediatric and adult Ph + ALL and Ph-like ALL Among adults with Ph + ALL, those with IKZF1 deletion had a higher cumulative incidence of relapse (69.1%) compared to those without IKZF1 deletion (40.4%; p = .0103). Consequently, Ph + ALL patients with IKZF1 deletion had a shorter DFS of only 10 months compared to 32 months for those wild-type patients (p = .01). In this population, there was no correlation between the type of IKZF1 deletion and prognosis, and OS was not different between the two groups [45]. However, among other clinically relevant factors such as age, white blood cell count at diagnosis, type of BCR-ABL1 transcript (p190 vs p210) and treatment protocols, only the presence of IKZF1 deletion constituted a negative prognostic factor on DFS but didn't reach statistical significance on OS in multivariate analysis. In this specific study, treatments included imatinib alone or in combination with cytotoxic chemotherapy backbone. The relative short follow-up at the time of the report may account for the fact that patients who relapsed may still be 10

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alive or those who relapsed may achieve a second remission with salvage therapy represent hypotheses to explain why OS was not significantly affected. A retrospective analysis of 66 adult Ph + ALL patients enrolled on the Chinese ChiCTR-TNRC-09000309 clinical trial, in which treatment consisted of imatinib and chemotherapy, identified three risk groups (low, intermediate and high risk) when combining IKZF1 deletion status with sustained early molecular response by MRD. Patients with double positive (high-risk group: IKZF1+/MRD+) experienced the worst outcome compared to that of the intermediate (IKZF1+/MRD- or IKZF1−/MRD+) and low-risk (IKZF1−/MRD-) groups [93]. These results highlight the clinical relevance of IKZF1 status to further refine prognostic stratification in Ph + ALL in addition to early treatment response measured by MRD. In children with Ph + ALL, IKZF1 deletion also emerges as an important prognostic factor. First, results from the EsPhALL group for childhood Ph + ALL have shown the unfavourable outcomes of children with Ph + ALL and IKZF1 deletion, irrespective of early clinical response and imatinib exposure. Among 191 children with Ph + ALL, 66% harbored an IKZF1 deletion. In the pre-imatinib era, the 4-year DFS for the IKZF1-deleted patients compared to wild-type patients was 30% vs 58% (p = .01) respectively. The presence of IKZF1 deletion still confers a poor prognosis in patients stratified in the good-risk arm based on early clinical response (52% for IKZF1-deleted vs 79% for wild-type; p = .03) and when treated with imatinib (56% for IKZF1deleted vs 75% for wild-type; p = .05) [59]. In AALL0622, a COG phase II trial testing the safety and efficacy of adding dasatinib to intensive chemotherapy on day 15 of induction for children with newly diagnosed Ph + ALL, 56.8% of tested patients harbour IKZF1 deletion. The latter was associated with inferior 5-year EFS (52% vs 82; p = .04) and OS (80% vs 100%; p = .04) compared to those with wild-type IKZF1. Interestingly, even among standard-risk Ph + ALL patients, defined by those who achieved early responses assessed by MRD (end-of-induction MRD < 1% and end-of-consolidation MRD < 0.01%), those with IKZF1 deletion had worse 5-year EFS compared to those without IKZF1 deletion (50% vs 83%; p = .04). The negative impact of IKZF1 deletion on outcome was not demonstrated among high-risk Ph + ALL patients in this cohort, which could be explained by the small number of patients in this subgroup analysis (n = 8) [94]. The adverse prognostic impact of IKZF1 deletion seen in Ph + ALL also phenocopies that of Ph-like ALL. Ph-like ALL with IKZF1-altered patients have the worst prognosis compared to those with wild-type IKZF1 and/or those without Ph-like ALL. This observation is consistent across different age groups; as the 5-year EFS for children with Ph-like ALL and IKZF1-altered was 48.6% compared to 71.7% for those with wild-type IKZF1 (p < .001); and 18.5% in young adults with both Phlike ALL and IKZF1-altered compared to 42.9% for those without IKZF1 alteration (p < .001) [8]. In the DFCI ALL Consortium Protocol 05–001 cohort, among 105 children with NCI HR B-ALL with available materials for genomic analysis, 15% of patients was found to harbour a kinase-activating fusion (P2RY8-CRLF2: n = 8; PAX5-JAK2: n = 4; ABL-class fusions: n = 4). 69% of those identified with fusion-positivity had a concomitant IKZF1 deletion. The 5-year EFS and OS were 44% and 62% respectively for patients with both fusion-positivity and IKZF1 deletion in comparison with 83% (p = .006) and 91% (p = .005) for those with neither. In univariate analysis, fusion-positivity and IKZF1 deletion were each associated with adverse outcomes; however, only IKZF1 deletion retained statistical significance in multivariable analysis (HR: 2.64; p = .019). Therefore, IKZF1 deletion represents an independent adverse prognostic factor, irrespective of the presence of a kinase-activating fusion [54]. Moreover, it should be recognized that the genomic heterogeneity of Ph-like ALL, the diagnostic complexity, the requirement of next-generation sequencing infrastructures, the lack of systematic screening and the relative rarity of each Ph-like genomically-defined subgroups, have hindered the development of prospective randomized trials for this patient population and constitute a disappointment in the era of precision medicine. International

collaborations are much needed to conduct well-designed trials for Phlike ALL to overcome the above challenges and thus, improving the outcomes of these patients. In the AEIOP-BFM ALL 2000 study for children with de novo B-ALL, Stanulla and colleagues described a new molecular profile associated with very-poor prognosis termed IKZF1plus. The latter is defined by the deletion of IKZF1 co-occurring with at least one of the following gene deletions: CDKN2A, homozygous CDKN2B, PAX5 or PAR1, and in the absence of ERG deletion. This distinct subgroup presented the worst prognosis and very high incidence of relapse when compared to their counterparts with IKZF1 deletion without co-deletion or with wild-type IKZF1. Specifically, the 5-year EFS for the above three groups was 53%, 79% and 87% respectively. The respective 5-year CIR for the same three groups was 44%, 11% and 10% respectively. Indeed, IKZF1plus confers the highest hazard ratio for relapse in multivariate analysis. Of importance, while MRD has previously been shown to be the strongest prognostic factor within this treatment protocol, the prognostic effect of IKZF1 alterations could be modulated by treatment response measured by MRD. In fact, the 5-year EFS for IKZF1plus –MRD SR (negative MRD at day 33 and day 78), IKZF1plus–MRD Intermediate-Risk (MRD positive by day 33 and negative by day 78) and IKZF1plus –MRD HR (MRD positive by day 78) was 94%, 40% and 30% respectively; while the corresponding 5-year CIR was 6%, 60% and 60% respectively. These results suggest that IKZF1plus has the potential to refine risk stratification in addition to MRD response [95,96]. The IKZF1plus subgroup in this study could encompass a high proportion of Ph-like ALL patients as both shared mutual adverse clinical and biologic features such as higher WBC at diagnosis, poor prednisone and MRD response, and higher frequency of the germline GATA3 variant rs3824662, formerly known to predispose to developing Ph-like ALL [97]. Furthermore, patients with IKZF1plus were retrieved in about half of adult Ph + ALL patients enrolled on 4 Gruppo Italiano Malattie EMatologiche dell'Adulto (GIMEMA) trials. The 3-year DFS and OS for IKZF1plus patients were both significantly inferior compared to those with sole IKZF1 deletions (24.9% vs 43.3%; p = .026 and 40.2% vs 62.6%; p = .02 respectively) [60]. 7. Refining risk stratification, current and future therapeutic strategies for IKZF1 alterations The above body of evidence amalgamates to highlight the clinical impact of IKZF1 alterations as an important adverse prognostic biomarker in pediatric and adult Ph-negative and Ph-driven ALL. Several pediatric collaborative groups have implemented the incorporation of IKZF1 gene status in current risk stratification algorithms. Upfront therapy intensification for IKZF1-mutated patients has been adopted as a therapeutic strategy for this now recognized as high-risk or very highrisk patient population. The DFCI, DCOG, UK, Malaysia-Singapore and AIEOP-BFM are actively screening for patients with IKZF1 deletion or IKZF1plus to non-randomly assign their most intensive post-induction chemotherapy regimens in order to improve outcomes (Fig. 2). Riskadapted therapy intensification for IKZF1-mutated patients has proven effective in a recent study from the Malaysia-Singapore ALL Consortium. The 5-year CIR for IKZF1-mutated patients in the recently completed clinical trial, MS2010 in which IKZF1 status was prospectively incorporated in risk assignment compared to the previous trial, MS2003, significantly reduced relapse rate from 30.4% to 13.5%. The 5-year OS for IKZF1-mutated patients in MS2010 increased to 91.6% vs 69.3% in MS2003 (p = .007) [98]. The role of hematopoietic stem cell transplant (HSCT) in post-remission intensification for IKZF1 status has not been investigated in prospective clinical trials. Nevertheless, frontline conventional chemotherapy intensification based solely on IKZF1 status raised concerns among other collaborative groups for several reasons. First, this approach may overtreat the favourable group of patients associated with ERG/DUX4 deregulation and frequent IKZF1 alterations and consequently lead to unnecessary toxicities. 11

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Fig. 2. Proposed algorithm upon identification of IKZF1 alterations for additional downstream testing and potential therapeutic interventions. Some examples of phase 3 clinical trials for newly diagnosed Ph + or Ph-like ALL patients are in blue for children and in orange for adults.

Moreover, certain groups felt that the prognostic effect of IKZF1 deletion alone (outside of Ph + and Ph-like ALL) was not profound enough to balance the risk of additional toxicities resulting from therapy intensification. Indeed, the adverse outcomes associated with IKZF1 alterations in Ph-negative B-ALL cohorts may be confounded by a large proportion of Ph-like ALL patients. There is a paucity of data assessing the prognostic impact of IKZF1 alterations alone independently from the Ph-like gene expression signature. Nevertheless, it is unclear whether more intensive chemotherapy regimen is the optimal therapeutic strategy in this context and will result in better outcomes for IKZF1mutated patients. Indeed, recent trials have shown that treatment intensification often translates into excessive toxicity rather than improved survival [99]. Therefore, other international consortia are seeking alternative therapeutic strategies to address the negative prognostic effect of IKZF1 deletion. The COG and other consortia do not assess directly the IKZF1 deletion status but will actively screen for the BCR-ABL1 oncoprotein or the Ph-like signature followed by downstream testing to identify the underlying kinase-activating alteration. This approach aims to combine the relevant TKI with intensive chemotherapy for those Ph + and Ph-like ALL with actionable genomic lesions, irrespective of IKZF1 status. Current precision medicine trials are actively enrolling Ph + and Ph-like patients to assess the safety and efficacy of such molecular-targeted approach. Increasing pre-clinical and clinical evidence speculated that TKI therapy may not be sufficient when IKZF1 co-occurred in Ph-positive and Ph-like ALL [45,46,59]. Churchman et al. performed a high-throughput drug screen in Arf−/ − /BCR-ABL1/IK6-expressing cells and identified retinoids as the most potent agents in inhibiting cellular aggregation. Indeed, retinoids completely reversed the aberrant adhesive and self-renewal phenotype of IKZF1-altered leukemic cells by inducing the expression of wild-type IKZF1 transcriptional program. Combined with dasatinib, the effect of retinoids was synergistic and frankly increased the survival of mice harbouring IKZF1-mutated BCR-ABL1 [38,39]. In parallel, focal adhesion kinase (FAK) inhibition in combination with dasatinib have shown exquisite response in inhibiting BCR-ABL1 leukemia models and can induce durable remission in vivo without recurrence, even after cessation of therapy [100]. The successful FAK inhibition stems from the

increased FAK pathway upregulation and overexpression of other adhesion molecules induced by IKZF1-mutated Ph + B-ALL cells; thus altering the bone marrow microenvironment and causing therapy resistance. Regarding specific drug compound, FAK inhibitor VS-4718 demonstrated potent activity against BCR-ABL1/IKZF1-mutant cells and PDX [38]. Other emerging IKZF1-directed therapeutic targets include CK2 and BCL6 inhibitors. In PDX models of high-risk leukemia, CK2 inhibition by CX-4945 results in anti-leukemic effect, prolonged survival, reduced leukemia progression and without significant toxic effects. This was thought to be mediated by enhancing of IKAROS regulation of the cell cycle and PI3K pathway [26]. Along the same line, Ge and colleagues explored the BCL6/BACH2 axis and its interaction with IKZF1 and CK2 inhibition. They demonstrated that IKZF1 deletion is associated with high BCL6/low BACH2 expression, and that CK2 inhibition rescues this function in B-ALL cells. They therefore hypothesized that a double hit to CK2 and BCL6 could be an interesting therapeutic target [101]. The above therapeutic targets remain in the realm of preclinical domain and may take time prior to being translated in clinical trials. More immediate therapeutic options including immunotherapeutic approaches should be explored in this patient population. Blinatumomab, a CD3/CD19 bispecific T-cell engager; inotuzumab ozogamicin, a CD22 monoclonal antibody conjugated to calicheamicin; and tisagenlecleucel, a CD19-directed chimeric antigen receptor (CAR) T-cell product, have all been FDA-approved for pediatric and adult relapsed/refractory B-ALL [102–104]. Although these studies have not specifically assessed for IKZF1 status, it is presumed that a high proportion of these relapsed/refractory B-ALL patients harbored IKZF1 alteration, especially among Ph + ALL and Ph-like ALL patients. Single agent blinatumomab or inozutumab have demonstrated antileukemic activity in relapsed/refractory Ph + ALL [103,105]. Combination of TKI and antibody-drug conjugates or bispecific monoclonal antibodies or CAR T cells for Ph + and Ph-like ALL patients are currently being explored [106,107]. Overall, immunotherapy alone or in combination with TKI represents a promising therapeutic avenue that is agnostic of sentinel genomic alterations and overcome the overlapping toxicity profile associated with cytotoxic chemotherapy. 12

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8. Conclusion

References

In conclusion, we reviewed the spectrum of IKZF1 alterations and their functional effects in ALL. We highlighted the predominance of IKZF1 deletions in kinase-driven leukemias such as Ph + and Ph-like ALL. The prognostic impact of IKZF1 alterations has been extensively reviewed among children and adults with Ph-negative and Ph-driven ALL. The vast majority of studies demonstrated that IKZF1 alterations consistently confer an independent adverse prognostic factor in patients with B-ALL. Hence, IKZF1 deletions emerge as a powerful biomarker to further refine risk stratification, especially in combination with early MRD response, which currently represents the strongest predictor of relapse in ALL. Therefore, this combinatorial approach using IKZF1 status and MRD may better select patients who could benefit from novel therapeutic approaches such as targeted therapy or immunotherapy. The optimal treatment strategies for IKZF1-mutated patients remain to be determined. International collaborations are essential to design wellpowered randomized precision medicine trials to study these rare and high-risk patient populations. The ongoing COG/EsPhALL clinical trial for Ph + ALL (NCT03007147), the first international prospective randomized clinical trial for pediatric Ph + ALL, laid the foundation to foster such precious collaborations that can extend to other rare ALL subtypes. Future research directions may further investigate the role of IKZF1 deletions as a surrogate biomarker involved in MRD monitoring for B-ALL. Such assays have been developed with comparable PCRbased MRD sensitivity but their interpretation and significance require additional validation in larger cohorts [108,109].

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Practice points

• IKZF1 alterations are prevalent in ALL subtypes associated with poor prognosis such as Ph + and Ph-like ALL. • When Ph-like ALL screening is not routinely available, the identifi• • •

cation of IKZF1 deletions should trigger the reflex to assess for the Ph-like gene expression signature and underlying genomic lesion that could be targeted by the relevant TKI. Although IKZF1 alterations confer a poor prognosis in the majority of ALL subtypes, it is important to exclude ERG/DUX4-deregulated ALL in which the high prevalence of IKZF1 alterations is not associated with unfavorable outcomes. The combination of IKZF1 status and MRD response may refine risk stratification in ALL. Clinical trials of targeted therapies in combination with chemotherapy and immunotherapeutic approaches for Ph + and Phlike ALL, irrespective of IKZF1 status, are ongoing.

Research agenda

• International collaborations to conduct well-designed, statistically •

powered randomized controlled trials incorporating IKZF1 status and MRD assessment in high-risk Ph-negative and Ph-driven ALL are much needed. Novel IKZF1-directed therapies such as retinoids or FAK inhibitors in combination with chemotherapy or immunotherapy should be investigated in early phase trials.

Declaration of Competing Interest Nothing to declare. Role of funding source No funding received. 13

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